Methods for analyzing rare circulating cells

ABSTRACT

The disclosure provides methods for analyzing rare circulating cells (RCCs) at cellular and molecular level following their detection in non-enriched blood samples, methods of this disclosure serve as diagnostic methods for several disease conditions, including cardiovascular diseases and cancer.

This application is a U.S. National Stage Application under 35 U.S.C. §371 of International Patent Application No. PCT/US2015/016499, filedFeb. 19, 2015, which claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/943,192, filed Feb. 21, 2014, the entirecontents of each of which are incorporated herein by reference.

The present disclosure relates generally to methods for the diagnosis ofdiseases such as cancer or cardiovascular diseases and, morespecifically, to methods for the molecular and cellular analysis of rarecirculating cells (RCCs), such as Circulating Tumor Cells (CTCs) orCirculating Endothelial Cells (CECs).

BACKGROUND

Certain types of rare cells circulating in the bloodstream (rarecirculating cells, RCCs) have recently emerged as highly promisingbiomarker candidates in a growing number of disease conditions. Forexample, Circulating Tumor Cells (CTCs) are considered promisingdiagnostic and prognostic markers for the monitoring of cancerprogression and anti-cancer treatment responses. Moreover, CirculatingEndothelial Cells (CECs) are considered promising diagnostic andprognostic markers in cardiovascular disease conditions, such as acutemyocardial infarction.

RCCs can be conveniently collected in blood samples (“liquid biopsy”),which enables repeated sampling throughout the course of a patient'sdisease progression or treatment regimen. Consequently, diagnosticmethods based on RCC detection, quantification and analysis enable thereal-time and personalized assessment of an individual patient'sdisease, which facilitates the design of personalized treatment plans.

However, the development of full biomarker utility of RCCs has beenhindered by the lack of assay technologies that can accurately androbustly identify and enumerate RCCs and also allow for the downstreamanalysis of RCC cell biology (e.g., gene expression, metabolic activity,protein localization, RNA localization) and RCC molecular biology (e.g.,genome, proteome, secretome, metabolome analysis). Especially theextremely low abundance of RCCs and the tremendous heterogeneity of RCCpopulations have posed substantial technical challenges for thedevelopment of reliable diagnostic assays.

Most existing RCC assay platforms lack the sensitivity and accuracy toallow for robust RCC identification and quantification. Moreover, thevast majority of RCC assay platforms do not allow for the detailedcellular or molecular analysis of RCCs once these cells have beenidentified and enumerated.

For example, many methods for RCC identification and quantification relyon flow cytometry (e.g., FACS) or immunocapture technologies (e.g.,CellSearch®). While flow cytometry generally enables cell sorting, itcannot robustly enumerate very small populations of cells, such as CTCsor CECs (˜1-10 CTCs/ml whole blood), in the presence of much moreabundant cell populations, such as the white blood cell population(WBC; >1 million CTCs/ml whole blood). Additionally, FACS-based methodsdo not allow for the indepth analysis of cell morphologies.

One prominent example of RCC immunocapture platforms is the CellSearch®platform, which has obtained FDA-approval for the monitoring ofmetastatic cancer patients. The CellSearch® CTC immunocapture assay hasrecently been adapted for CEC detection (see, e.g., Damani, et al.,2012, Sci. Tansl. Med. 4, 126 ra33). However, CellSearch® and relatedimmunocapture platforms require an initial immunomagnetic bead-basedcapture step that targets a single biomarker to enrich the very rareRCCs in a sample prior to an attempted identification andquantification. It is this initial, targeted enrichment step that renderan unbiased multi-parametric analysis and classification ofheterogeneous RCC populations impossible and that prevents any analysisfrom reaching much beyond the analysis of the single biomarker used forcell capture. Moreover, RCC-targeted immunocapture assays are oftenplagued by a lack of assay sensitivity and specificity.

Due to the limitations of many existing assay technologies, the RCClevels reported for human blood samples vary greatly across theliterature, even though substantial assay optimization andstandardization efforts were made. This variability in RCC assay resultssignificantly impedes the further development of RCCs as clinicallyuseful biomarkers. Another caveat of most existing RCC assaytechnologies is the limited amount of diagnostically relevantinformation that is commonly obtained. Typical RCC assays may deliverRCC counts and describe general morphological features of a cell (e.g.,cell size, size distributions across a cell population), but current RCCassays typically do not provide a diagnostically meaningful profile ofRCC cell biology (e.g., regarding the energy metabolism of cancer cellsor the presence of apoptotic bodies) or RCC molecular biology (e.g.,presence of genetic abnormalities, including gene fusions, aneuploidy,loss of chromosomal regions, specific oncogene mutations or oncogeneexpression levels). Thus, new approaches are needed to accuratelyidentify, enumerate and analyze RCCs.

Recently, a high-definition (HD) immunofluorescence assay platform hasbeen developed, which enables the reliable identification andenumeration of RCCs in the presence of much more abundant cell types.HD-RCC assays are generally based on the side-by-side comparison of rarecells (e.g., CTCs or CECs) and abundant cells (e.g., WBCs) innon-enriched samples (e.g., blood samples) with respect to certainimmunofluorescent and morphological characteristics. Most notably,HD-CTC and HD-CEC assays have proven to enable the highly sensitive,highly accurate, and highly robust detection and quantification of CTCsand CECs.

While current HD-RCC assay protocols enable accurate cell identificationand cell counting, robust protocols for the subsequent downstreamanalysis of RCC cell biology or RCC molecular biology are still largelylacking today. Nevertheless, it is widely expected that a deeperunderstanding of RCC biology will promote the development of meaningfuldisease diagnostics and efficacious treatments. For example, it isexpected that a better understanding of CTC biology will promote thedevelopment of next-generation anti-cancer treatments that target CTCsand thereby help suppress tumor metastasis. Moreover, it is expectedthat the detection of certain molecular characteristics of CTCs willhave immediate diagnostic value (e.g., detection of BRCA-1/2 mutations)and aid in the personalized tailoring of anti-cancer treatment regimensto each patient (e.g., treatment with PARP inhibitors).

Thus, there exists a need for methods enabling the cellular andmolecular analysis of RCCs following RCC detection. The presentdisclosure addresses this need by providing methods for the analysis ofRCCs in non-enriched patient samples. Related advantages are provided aswell.

SUMMARY

The present disclosure provides methods for further characterizing CTCsfollowing their identification in a non-enriched biological sample

In one aspect, the disclosure provides a method for analyzing rarecirculating cells (RCCs) in a non-enriched blood sample, including: (a)detecting RCCs in the non-enriched blood sample, including i)determining presence or absence of one or more immunofluorescent RCCdetection markers in nucleated cells in the non-enriched blood sample,and ii) assessing the morphology of the nucleated cells, wherein RCCsare detected among the nucleated cells based on a combination ofdistinct immunofluorescent staining and morphological characteristics;(b) quenching the immunofluorescence of the one or moreimmunofluorescent RCC detection markers including contacting the RCCswith a quenching buffer, wherein the immunofluorescence is quenched bymore than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.99%; and (c)analyzing the detected RCCs, comprising determining presence or absenceof one or more fluorescent RCC analysis markers.

In some embodiments, the fluorescent RCC analysis markers arefluorescence in situ hybridization (FISH) markers.

In some embodiments, more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of RCCs detected in (a) areretained in (c).

In some embodiments, the fluorescent RCC analysis markers are positivecontrol markers. In some embodiments, the positive control markers arepresent in more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99% of RCCs analyzed in (c). In someembodiments, the positive control markers are chromosomal markers. Insome embodiments, the positive control markers are centromer markers ortelomere markers.

In some embodiments, the fluorescent RCC analysis markers are geneticmutations selected from the group consisting of gene translocation, geneamplification gene deletion, gene aneuploidy and chromosomal aneuploidy.

In some embodiments, analyzing the detected RCCs further comprisesassessing the morphology of the detected RCCs.

In some embodiments, the RCCs are circulating tumor cells (CTC). In someembodiments, the RCCs are a circulating epithelial cell (CEC). In someembodiments, the RCCs are CTC mimics. In some embodiments, the RCCs areCTC candidates.

In some embodiments, the quenching buffer comprises a chaotropic agent.In some embodiments, the concentration of the chaotropic agent is atleast 2M, 3M or 4M. In some embodiments, the quenching buffer comprisesa chaotropic salt. In some embodiments, the quenching buffer comprisesguanidine or a guanidinium salt. In some embodiments, the quenchingbuffer comprises guanidinium thiocyanate (guanidine thiocyanate) orguanidinium chloride (guanidine hydrochloride).

In some embodiments, the method is performed by fluorescent scanningmicroscopy.

In some embodiments, the microscopy provides a field of view comprisingmore than 2, 5, 10, 20, 30, 40 or 50 RCCs, wherein each RCC issurrounded by more than 10, 50, 100, 150 or 200 WBCs.

In some embodiments, determining presence or absence of theimmunofluorescent RCC detection markers comprises comparing the distinctimmunofluorescent staining of RCCs with the distinct immunofluorescentstaining of WBCs.

In some embodiments, determining presence or absence of the fluorescentRCC analysis markers comprises comparing the distinct fluorescentstaining of RCCs with the distinct fluorescent staining of WBCs.

In some embodiments, assessing the morphology of the nucleated cellscomprises comparing the morphological characteristics of RCCs with themorphological characteristics of surrounding WBCs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images illustrating the relative quenching effects ofGuanidinium Thiocyanate Buffer (GdnSCN) and Western Blot StrippingBuffer (WBSB) on CTC immunofluorescence staining Images in the top rowshow exemplary nuclear staining (DAPI) of WBCs and CTCs in anon-enriched blood sample. Images in the bottom row show exemplaryresidual cytokeratin (CK) immunofluorescence staining of CTCs aftertreatment with GdnSCN (4M, 10 minutes; first and second columns) orafter treatment with WBSB for 5 minutes or 10 minutes (third, fourth andfifth columns).

FIG. 2 shows images further illustrating the relative quenching effectsof Guanidinium Thiocyanate Buffer (GdnSCN, 4M) and Western BlotStripping Buffer (WBSB) on CTC immunofluorescence staining. Images inthe top row show exemplary nuclear staining (DAPI) of WBCs and CTCs in anon-enriched blood sample. Images in the bottom row show exemplaryresidual cytokeratin (CK) immunofluorescence staining of CTCs aftertreatment with GdnSCN (first column) at room temperature (RT) or aftertreatment with WBSB at room temperature (second column) or at 37° C.(third column).

FIG. 3 shows images illustrating the relative quenching effects ofGuanidinium Thiocyanate Buffer (GdnSCN, 4M) and Sodium ThiocyanateBuffer (NaSCN, 4M) on CTC immunofluorescence staining. Images in the toprow show exemplary nuclear staining (DAPI) of WBCs and CTCs in anon-enriched blood sample. Images in the bottom row show exemplaryresidual cytokeratin (CK) immunofluorescence staining of CTCs aftertreatment with GdnSCN (left column) or after treatment with NaSCN (rightcolumn).

FIG. 4 shows images illustrating the concentration-dependent quenchingeffect of Guanidinium Thiocyanate Buffer (GdnSCN). Images in the top rowshow exemplary nuclear staining (DAPI) of WBCs and CTCs in anon-enriched blood sample. Images in the bottom row show exemplaryresidual cytokeratin (CK) immunofluorescence staining of CTCs aftertreatment with GdnSCN at concentrations of 4 M, 3 M, 2 M or 1M.

FIG. 5 shows images illustrating the relative effects of a GuanidiniumThiocyanate Buffer (GdnSCN, 4M), an acidic Glycine Buffer (pH 2) and anacidic Glycine/SDS Buffer (Pre-Fix) on CTC immunofluorescent stainingand cell viability. Images in the top row show exemplary nuclearstaining (DAPI) of WBCs and CTCs in a non-enriched blood sample. Imagesin the bottom row show exemplary residual cytokeratin (CK)immunofluorescence staining of CTCs after treatment with GdnSCN (4M,left column), Glycine (pH 2, 50° C.; center column) orSDS(1%)/Glycine(pH2.2) (pre-fixed with 1% formaldehyde; right column).

FIG. 6 shows images illustrating the relative effects of a GuanidiniumThiocyanate Buffer (GdnSCN, 4M), a neutral Glycine Buffer (pH 7) and abasic Glycine Buffer (pH 10) on CTC immunofluorescent staining and cellviability. Images in the top row show exemplary nuclear staining (DAPI)of WBCs and CTCs in a non-enriched blood sample. Images in the bottomrow show exemplary residual cytokeratin (CK) immunofluorescence stainingof CTCs after treatment with GdnSCN (left column), Glycine (pH 2; centercolumn) or Glycine/SDS (Pre-Fix; right column).

FIG. 7 shows images illustrating the relative effects of a GuanidiniumThiocyanate Buffer (GdnSCN, 4M) and an SDS(1%)/Glycine Buffer (pH 2.2)on CTC immunofluorescent staining and cell viability. Images in the toprow show exemplary nuclear staining (DAPI) of WBCs and CTCs in anon-enriched blood sample. Images in the bottom row show exemplaryresidual cytokeratin (CK) immunofluorescence staining of CTCs aftertreatment with GdnSCN (left column) or SDS (1%)/Glycine (pH2.2) (roomtemperature; right column).

FIG. 8 shows images illustrating exemplary results of a FISH experimentconducted on a non-enriched blood sample, following the identificationof CTCs in an HD-CTC assay of this disclosure and the subsequentquenching of CK-immunofluorescence with 4M GdnSCN quenching buffers. Reddots represent signals of the FISH-control probe (chromosome 10centromere probe; Texas Red channel); green dots represent signals ofFISH-probes targeting a gene of interest (PTEN; FITC channel).

DETAILED DESCRIPTION

The present disclosure is based, in part, on the discovery that RCCs canbe subjected to further analysis of their cellular or molecularcharacteristics after they were identified, classified (e.g., as CTCs,CTC mimics, CTC candidates or CECs) and quantified in a non-enrichedbiological sample. Specifically, the present disclosure is based, inpart, on the discovery that RCCs, such as CTCs, CTC mimics, CTCcandidates or CECs, can be subjected to further analysis after they wereidentified, classified and quantified in a non-enriched biologicalsample using an HD-RCC assay.

In certain embodiments, HD-RCC assays include, inter alia, detectingRCCs in non-enriched blood samples by determining presence or absence ofcertain immunofluorescent RCC detection markers. For example, thedetection of CTCs can include determining the presence or absence of theimmunofluorescent marker cytokeratin. In another example, the detectionof CECs can include determining the presence or absence of theimmunofluorescent detection marker Von Willebrand factor (vWF).

The present disclosure is further based, in part, on the discovery thatin HD-RCC assays, the RCCs can be further analyzed by determiningpresence or absence of certain immunofluorescent RCC analysis markers.For example, in some embodiments, RCCs can be further analyzed bydetermining presence or absence of oncogene mutations or of elevatedoncogene expression levels. In some embodiments, determining presence orabsence of RCC analysis markers includes in situ fluorescencehybridization (FISH).

The present disclosure is further based, in part, on the discovery that,in some embodiments, determining the presence or absence of animmunofluorescent RCC analysis marker requires quenching theimmunofluorescence of an immunofluorescent RCC detection marker.

The present disclosure is further based, in part, on the discovery thatit is technically very challenging to effectively quench theimmunofluorescence of an immunofluorescent RCC detection marker while,at the same time, retaining RCCs for subsequent analysis and whilemaintaining the RCCs in a condition that allows for the subsequentdetection of immunofluorescent RCC detection markers. For example, itwas found that many quenching buffers and many assay buffers (e.g., FISHbuffers) that are commonly used by skilled artisans either did notsufficiently quench the immunofluorescence of RCC detection marker (see,e.g., FIG. 2; Western Blot Stripping Buffer, WBSB) or resulted in theloss of previously detected RCCs or rendered the remaining RCCs in acondition that did not allow for the subsequent detection ofimmunofluorescent RCC analysis markers or rendered the downstreamanalysis of RCCs otherwise impossible (see, e.g., FIG. 5, Glycine Buffer(pH 2)).

The present disclosure is further based, in part, on the surprisingdiscovery that the immunofluorescence of an RCC detection marker can infact be quenched while retaining a substantial number of RCCs forfurther analysis.

The present disclosure is further based, in part, on the surprisingdiscovery that the presence of certain immunofluorescent RCC analysismarkers can be detected in a substantial number of RCCs after theimmunofluorescence of an immunofluorescent RCC detection marker has beenquenched.

The present disclosure is further based, in part, on the surprisingdiscovery that highly effective quenching buffers included buffers thata skilled artisan would not expect to yield high quality results whenapplied in a method of this disclosure, for example and without wishingto be bound by theory, because the buffers would be considered highlystressful or disruptive on cells and resulting in either cell loss orloss of RCC analysis marker signals (e.g., buffers containing chaotropicreagents).

The present disclosure is further based, in part, on the surprisingdiscovery that the morphological characteristics of RCCs as well asother nucleated cells in the sample (e.g., white blood cells, WBCs) canbe largely maintained after treatment of RCCs with effective quenchingbuffers that contain chaotropic agents, such as guanidinium thiocyanateand the like.

A fundamental aspect of the present disclosure is the robustness of thedisclosed methods. The rare event detection (RED) disclosed herein withregard to RCCs is based on a direct analysis of a non-enriched cellpopulation that encompasses the identification of rare events in thecontext of the surrounding non-rare events. Identification of the rareevents according to the disclosed methods inherently identifies thesurrounding events as non-rare events. Taking into account thesurrounding non-rare events and determining the averages for non-rareevents, for example, average cell size of non-rare events, allows forcalibration of the detection method by removing noise. The result is arobustness of the disclosed methods that cannot be achieved with methodsthat are not based on direct analysis but that instead compare enrichedpopulations with inherently distorted contextual comparisons of rareevents.

The disclosure provides methods for further analyzing RCCs (e.g., CTCs,CTC mimics, CTC candidates or CECs) after they were identified innon-enriched blood samples. One major advantage of the presentdisclosure is the combination of a highly accurate and sensitive methodfor identifying, classifying and quantifying RCCs with downstreammethods for analyzing RCCs with respect to their cell biology andmolecular biology traits. In certain aspects the downstream analysisRCCs includes the analysis of disease and biomarkers, e.g., thedetection of oncogene mutations (e.g., BRCA-1/2 mutations) in CTCs orthe detection of aberrant oncogene expression levels (e.g., HER2expression levels) in CTCs. It is widely expected that by combining anaccurate RCC quantification with the downstream detection of diseasemarkers or biomarkers the diagnostic and prognostic value of RCCs willbe potentiated.

As a result, the present disclosure is of particular benefit, forexample, to human patients. Specifically, cancer patients will benefitfrom the improved diagnosis of their disease. For example, the methodsof this disclosure will improve the diagnosis of neoplastic progressionor recurrence and allow for the real-time analysis of a tumor'sdevelopment at the molecular and cellular level. An improvedunderstanding of a patient's tumor biology is generally expected tofacilitate the personalization of treatment regimens and improvetreatment outcomes.

Provided herein are methods for analyzing rare circulating cells (RCCs)in a non-enriched biological sample, including: (a) detecting RCCs inthe non-enriched biological sample, including i) determining presence orabsence of one or more RCC detection markers in nucleated cells in thenon-enriched biological sample, and ii) assessing the morphology of thenucleated cells, wherein RCCs are detected among the nucleated cellsbased on a combination of distinct detection marker staining andmorphological characteristics; (b) quenching the staining of the one ormore RCC detection markers comprising contacting the RCCs with aquenching buffer, wherein the staining is quenched by more than 50%,60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.99%; and (c) analyzing thedetected RCCs, comprising determining presence or absence of one or moreRCC analysis probes.

Further provided herein are methods for analyzing rare circulating cells(RCCs) in a non-enriched blood sample, including: (a) detecting RCCs inthe non-enriched blood sample, including i) determining presence orabsence of one or more immunofluorescent RCC detection markers innucleated cells in the non-enriched blood sample, and ii) assessing themorphology of the nucleated cells, wherein RCCs are detected among thenucleated cells based on a combination of distinct immunofluorescentstaining and morphological characteristics; (b) quenching theimmunofluorescence of the one or more immunofluorescent RCC detectionmarkers comprising contacting the RCCs with a quenching buffer, whereinthe immunofluorescence is quenched by more than 50%, 60%, 70%, 80%, 90%,95%, 99%, 99.9% or 99.99%; and (c) analyzing the detected RCCs,comprising determining presence or absence of one or more fluorescentRCC analysis markers.

It must be noted that, as used in this specification and the appendedclaims, the term “about,” particularly in reference to a given quantity,is meant to encompass deviations of plus or minus five percent.

As used in this application, including the appended claims, the singularforms “a,” “an,” and “the” include plural references, unless the contentclearly dictates otherwise, and are used interchangeably with “at leastone” and “one or more.”

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but can include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

The biological samples of this disclosure can be any sample suspected tocontain RCCs (e.g., CTCs, CTC candidates, CTC mimics, CECs), includingsolid tissue samples, such as bone marrow, and liquid samples, such aswhole blood, plasma, amniotic fluid, pleural fluid, peritoneal fluid,central spinal fluid, urine, saliva and bronchial washes. In someembodiments, the biological sample is a blood sample. As will beappreciated by those skilled in the art, a biological sample can includeany fraction or component of blood, without limitation, T-cells,monocytes, neutrophils, erythrocytes, platelets and microvesicles suchas exosomes and exosome-like vesicles.

The biological samples of this disclosure can be obtained from anyorganism, including mammals such as humans, primates (e.g., monkeys,chimpanzees, orangutans, and gorillas), cats, dogs, rabbits, farmanimals (e.g., cows, horses, goats, sheep, pigs), and rodents (e.g.,mice, rats, hamsters, and guinea pigs).

It is noted that, as used herein, the terms “organism,” “individual,”“subject,” or “patient” are used as synonyms and interchangeably.

The organisms of this disclosure include healthy organisms and diseasedorganisms.

Diseased organisms can suffer from any disease associated with aberrantRCC levels. The term “aberrant RCC levels”, as used herein, refers toRCC levels in a sample that significantly deviate from the median RCClevels found in a population of healthy organisms. In some embodiments,the aberrant RCC levels are higher than the median RCC levels. In someembodiments, the aberrant RCC levels are lower than the median RCClevels.

In some embodiments, the healthy organisms have never suffered from acertain disease. In some embodiments, the healthy organisms werepreviously diseased. In some embodiments, the healthy organisms areundergoing a routine medical checkup. In some embodiments, the healthyorganisms are members of a control group in a clinical trial. In someembodiments, the healthy organisms are at risk of contracting a disease,as determined by the presence of certain risk factors that are wellknown in the art. Such risk factors include, without limitation, agenetic predisposition, a personal disease history, a familial diseasehistory, a lifestyle factor, an environmental factor, a diagnosticindicator and the like.

In some embodiments, the organism is at risk of suffering frommyocardial infarction or another cardiovascular disease. In someembodiments, the organism has a genetic predisposition for developing acardiovascular disease (e.g., resulting in high cholesterol levels,diabetes, obesity) or a family history of cardiovascular diseases. Insome embodiments, the organism is subject to certain lifestyle factorspromoting the development of cardiovascular disease (e.g., cigarettesmoking, low exercise, high body/mass index, high fat “western” diet) orshows clinical disease manifestations of cardiovascular disease (e.g.,atherosclerotic plaques, hypertension, prior medical history of thepatient, chest pain, numbness in left arm). In some embodiments, theorganism is a patient who is receiving a clinical workup (e.g.,electrocardiogram (ECG), blood work) to diagnose a heart attack or therisk of a heart attack. In some embodiments, a heart attack is expectedto be imminent (e.g., a heart attack expected to occur within one weekfrom the time of the clinical workup). In some embodiments, the organismis a patient having elevated blood levels of troponin relative to normalcontrols.

In some embodiments, the organism is at risk of developing a cancer. Insome embodiments, the organism has a genetic predisposition for cancer(e.g., BRCA 1 or BRCA 2 mutations) or a family history of cancer. Insome embodiments, the organism was exposed to carcinogens (e.g., acigarette smoke, exhaust fumes, smog, asbestos, environmental pollution,toxins and the like).

In some embodiments, the diseased organism suffers from a cardiovasculardisease such as myocardial infarction (MI; e.g., acute myocardialinfarction (MI) or stable coronary artery disease (CAD)) or stroke. Insome embodiments, the diseased organism suffers from a metabolicsyndrome (e.g., diabetes, obesity).

In some embodiments, the diseased organism suffers from cancer. Thecancers of this disclosure typically form a solid tumor. The tumor caninclude a primary tumor and a metastatic tumor. The tumor can bevascularized. The cancers can be at least partly responsive to therapy(e.g., surgery, chemotherapy, radiation therapy) or unresponsive totherapy. The cancers can be resistant to one or more anti-cancertreatments (e.g., resistant to specific chemotherapy regimens). Thecancers can include cancers of all stages, e.g., stage I, stage II,stage III, or stage IV cancers.

The cancers of this disclosure include, without limitation, lung cancer(e.g., small-cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), including, e.g., adenocarcinomas or lung carcinoid tumor), skincancer, colon cancer, renal cancer, liver cancer, pancreatic cancer,thyroid cancer, bladder cancer, gall bladder cancer, brain cancer (e.g.,glioma, glioblastoma, medulloblastoma, neuroblastoma), breast cancer,ovarian cancer, endometrial cancer, prostate cancer, testicular cancerand lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, T-celllymphoma, B-cell lymphoma).

In some embodiments, the diseased organism is treatment naïve. In someembodiments, the diseased organism has received a treatment prior tosample collection. In some embodiments the diseased organism isundergoing treatment at the time of sample collection. Treatments caninclude, without limitation, drug treatments, radiation treatments,surgery, and the like.

In some embodiments, the treatment includes a drug treatment (e.g., betablockers, anti-coagulants (e.g., aspirin, plavix), nitro, heparin,morphine, statins, insulin, chemotherapy, VEGF antagonists, EGFRantagonists, HER2 antagonists, kinase inhibitors). In some embodiments,the treatment includes surgery (e.g., endarterectomy, tumor excision).In some embodiments, the treatment includes radiation therapy. In someembodiments, the treatment includes a combination of treatements (e.g.,a combination of two or more drug treatments, a combination of a drugtreatment with a radiation treatment).

In some embodiments, the organism is an animal model. In someembodiments, the organism is an animal model for a cardiovasculardisease. In some embodiments, the organism is an animal model forcancer, including, without limitation, a xenograft mouse model, atransgenic mouse carrying a transgenic oncogene, a knockout mouselacking a proapoptotic gene and others. A person of ordinary skillunderstands that animal models (in mice or other organisms) are wellknown in the art for a series of disease conditions.

In some embodiments, the blood sample was obtained from a patient. Insome embodiments, the patient received a treatment for a period of time(e.g., for more than 1 day, 1 week, 1 month, 3 months, 6 months, 9months, 1 year, 2 years, 3 years, 4 years, 5 years). In some embodimentsthe blood sample is a plurality of blood samples. In some embodimentsthe plurality of blood samples were collected over a period of time. Insome embodiments, at least one blood sample of the plurality of bloodsamples was collected before the patient received a treatment for aperiod of time. In some embodiments, at least one blood sample of theplurality of blood samples was obtained when the patient was treatmentnaïve. In some embodiments, at least one blood sample of the pluralityof blood samples was obtained from a patient during the period of timewhen the patient received a treatment. In some embodiments, at least oneblood sample of the plurality of blood samples was obtained before thepatient received a treatment for a period of time and at least one bloodsample of the plurality of blood samples was obtained during the periodof time when the received the treatment. In some embodiments, a firstblood sample was obtained at a first time during the period of time whenthe patient received a treatment and a second blood sample was obtainedat a second time during the period of time when the patient received thetreatment. In some embodiments the first time and the second time wereseparated by a period of time of more than 1 day, 1 week, 2 weeks, 1month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years,4 years or 5 years.

In some embodiments, the blood sample was obtained from a non-small celllung cancer (NSCLC) patient. In some embodiments, the blood sample wasobtained from a MI patient.

In some embodiments, the methods further include the initial step ofobtaining a blood sample from a patient.

The samples of this disclosure can each contain a plurality of cellpopulations and cell subpopulation that are distinguishable by methodswell known in the art (e.g., FACS, immunohistochemistry). For example, ablood sample can contain populations of non-nucleated cells, such aserythrocytes (e.g., 4-5 million/μl) or platelets (150,000-400,000cells/μl), and populations of nucleated cells such as white blood cells(WBCs, e.g., 4,500-10,000 cells/μl), CECs or CTCs (circulating tumorcells; e.g., 2-800 cells/μl). WBCs can contain cellular subpopulationsof, e.g., neutrophils (2,500-8,000 cells/μl), lymphocytes (1,000-4,000cells/μl), monocytes (100-700 cells/μl), eosinophils (50-500 cells/μl),basophils (25-100 cells/μl) and the like. The samples of this disclosureare non-enriched samples, i.e., they are not enriched for any specificpopulation or subpopulation of nucleated cells. For example,non-enriched blood samples are not enriched for any WBCs, B-cells,T-cells, NK-cells, monocytes, or the like. Specifically, the bloodsamples of this disclosure are not enriched for any RCC, including CTCs,CTC mimics, CECs or the like.

The samples of this disclosure can be obtained by any applicable methodknown to a person of skill, including, e.g., by solid tissue biopsy orby fluid biopsy (see, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9016003; Nieva J. et al., 2012, Phys. Biol. 9 016004). A blood sample canbe extracted from any source known to include blood cells or componentsthereof, such as venous, arterial, peripheral, tissue, cord and thelike. The sample can be processed using well known and routine clinicalmethods (e.g., procedures for drawing and processing whole blood). Insome embodiments, a blood sample is drawn into anti-coagulant bloodcollection tubes (BCT), which can contain EDTA or Streck Cell-Free DNA™.In other embodiments, a blood sample is drawn into CellSave® tubes(Veridex). A blood sample can be stored for up to 12 hours, 24 hours, 36hours, 48 hours, 60 hours, 72 hours, 86 hours, 96 hours, 108 hours or120 hours or longer before further processing.

In some embodiments, the methods of this disclosure comprise obtaining awhite blood cell (WBC) count for the blood sample. In certainembodiments, the WBC count may be obtained by using a HemoCure® WBCdevice (Hemocure, Ängelholm, Sweden).

In some embodiments, the methods of this disclosure comprise a step oflysing erythrocytes in the blood sample. In some embodiments, theerythrocytes are lysed, e.g., by adding an ammonium chloride solution tothe blood sample. In certain embodiments, a blood sample is subjected tocentrifugation following erythrocyte lysis and nucleated cells areresuspended, e.g., in a PBS solution.

In some embodiments, nucleated cells from a sample, such as a bloodsample, are deposited as a monolayer on a planar support. The planarsupport can be of any material, e.g., any fluorescently clear material,any material conducive to cell attachment, any material conducive to theeasy removal of cell debris, any material having a thickness of <100 μm.In some embodiments, the material is a film. In some embodiments thematerial is a glass slide. The glass slide can be coated to allowmaximal retention of live cells (See, e.g., Marrinucci D. et al., 2012,Phys. Biol. 9 016003). In some embodiments, about 0.5 million, 1million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4million, 4.5 million, or 5 million nucleated cells are deposited ontothe glass slide. In some embodiments, the methods of this disclosurecomprise an initial step of depositing nucleated cells from the bloodsample as a monolayer on a glass slide. In certain embodiments, themethod comprises depositing about 3 million cells onto a glass slide. Insome embodiments, the WBC count is used to determine the amount of bloodrequired to plate a consistent loading volume of nucleated cells perglass slide.

In some embodiments, the methods of this disclosure comprise an initialstep of identifying nucleated cells in the non-enriched blood sample. Insome embodiments, the nucleated cells are identified with a fluorescentstain. In certain embodiments, the fluorescent stain comprises a nucleicacid specific stain. In certain embodiments, the fluorescent stain isdiamidino-2-phenylindole (DAPI).

The term “rare cell”, as used herein, refers to a cell that has anabundance of less than 1:1,000 in a cell population, e.g., an abundanceof less than 1:5,000, 1:10,000, 1:30,000, 1:50:000, 1:100,000,1:300,000, 1:500,000, or 1:1,000,000. In some embodiments, the rare cellhas an abundance of 1:50:000 to 1:100,000 in the cell population.

The term “sample cell”, as used herein, refers to any cell in a samplethat is not a rare cell. For example, sample cells in a blood sampleinclude WBCs.

In some embodiments, the rare cells of this disclosure are rarecirculating cells (RCCs). In some embodiments, the RCCs are circulatingin the blood stream of an organism. In some embodiments, the RCC is acirculating tumor cell (CTC). In some embodiments, the RCC is acirculating epithelial cell (CEC). In some embodiments, the RCC is a CTCmimic. In some embodiments, the RCC is a CTC candidate.

The Circulating Tumor Cells (CTCs) of this disclosure are tumor cellsthat are circulating in the bloodstream of an organism.

The Circulating Endothelial Cells (CECs) of this disclosure areendothelial cells that are circulating in the bloodstream of anorganism.

The term “CTC mimic”, as used herein, refers to a cell that, whilesharing one or more biomarkers, morphological characteristics, or acombination thereof, with a CTC, is not a CTC. In some embodiments, aCTC mimic is a CEC.

The term “CTC candidate”, as used herein, refers to a cell that isdetected based on the presence of a biomarker or a morphologicalcharacteristic, or combination thereof, that is shared between CTCmimics and CTCs. A CTC candidate can be a CTC or a CTC mimic. A CTCcandidate can be identified as a CTC or a CTC mimic based on thedetection of further biomarkers, further morphological characteristics,or combinations thereof that are characteristic of a CTC or another RCC.

The RCCs of this disclosure are detected among the nucleated cells of asample based on a combination of distinct biomarkers and morphologicalcharacteristics.

The term “CEC detection marker”, as used herein, refers to a biomarkerthat can be used to detect CECs, but not certain other RCCs (e.g., CTCs)or certain sample cells (e.g., WBCs). In some embodiments, the CECmarker is present in CECs, CTC mimics and CTC candidates and absent inCTCs and WBCs.

CEC detection markers include, without limitation, any biomarker that isspecific for endothelial cells (e.g., cluster of differentiation (CD)146, Von Willebrand factor (vWF), CD 31, CD 34, or CD 105).

The term “CTC detection marker”, as used herein, refers to a biomarkerthat can be used to detect CTCs, but not certain other RCCs (e.g., CECs)or certain sample cells (e.g., WBCs). In some embodiments, the CTCdetection marker is present in CTCs, CTC candidates and CTC mimics andabsent in CECs and WBCs.

CTC detection markers include, without limitation, any cancer-specificbiomarker. Cancer-specific biomarkers can include, for example,biomarkers that are specific for a given cancer-type of interest (e.g.,non-small cell lung cancer, NSCLC), a clinical cancer-stage of interest(e.g., stage IV), or a cancer cell property of interest (e.g., energymetabolism, epithelial-mesenchymal transition). Additionally,cancer-specific biomarkers can include more general cancer markers, suchas cancer markers that are present in several cancer-types, but not innormal cells, or cancer markers that generally signal the malignanttransformation of a cell. A person of skill will recognize that manyspecific and general cancer-specific biomarkers are known in the art.

CTC detection markers include, without limitation, anaplastic lymphomakinase (ALK), androgen receptor (AR), Axl, cMET, cytokeratins 1, 4, 5,6, 7, 8, 10, 13, 18 or 19; CD 31, CD 99, CD 117, chromatogranin, desmin,E-cadherin, epidermal growth factor receptor (EGFR), epithelial celladhesion molecule (EpCAM), epithelial membrane antigen (EMA), grosscystic disease fluid protein (GCDFP-15), HMB-45, inhibin, MART-1, MCM2,Myo D1, muscle-specific actin (MSA), N-cadherin, neurofilament,neuron-specific enolase (NSE), p63, placental alkaline phosphatase(PLAP), prostate specific membrane antigen (PSMA), S100 protein, smoothmuscle actin (SMA), synaptophysin, thyroid transcription factor-1(TTF-1), tumor M2-PK (i.e., pyruvate kinase isoenzyme type M2), vimentinand more.

The term “RCC detection marker”, as used herein, refers to a biomarkerthat is present in a RCC of interest, but not in a sample cell. In someembodiments, the RCC marker is present in one type of RCCs (e.g., a CECmarker that is only present in CECs). In some embodiments, the RCCmarker is present in more than one type of RCCs (e.g., a CTC marker thatis present in CTCs and CTC mimics). The RCC detection markers of thisdisclosure can be used to detect RCCs, but not sample cells, such asWBCs. RCC detection markers include, for example, CTC markers, CECmarkers and the like.

The term “sample cell detection marker”, as used herein, refers to anybiomarker that is present in at least one sample cell, but that is notpresent in an RCC of interest. In some embodiments, the sample celldetection marker is present in at least one cell-type in the sample andabsent in CECs, CTCs, CTC candidates and CTC mimics. The sample celldetection markers of this disclosure are present in a sample cell thatis more abundant than CECs, CTCs, CTC candidates, or CTC mimics. In someembodiments, the sample cell detection marker is present in a WBC andabsent in CECs, CTCs, CTC candidates and CTC mimics. In someembodiments, the sample cell detection marker is CD 45. In someembodiments the methods include determining presence or absence of asample cell detection marker.

The term “biomarker,” as used herein, refers to a biological molecule,or a fragment of a biological molecule, the change and/or the detectionof which can be correlated with the identity of an RCC or with aparticular physical condition or state of an RCC. In some embodiments,the biomarkers are detection markers. In some embodiments, thebiomarkers are analysis markers.

The term “detection marker”, as used herein, refers to a biomarker thatis used to identify a cell as belonging to a certain cell-type ofinterest, e.g., a CTC, CEC or WBC. Detection markers can be used todifferentiate one cell type from another cell type (e.g., differentiatea CTC mimic from a CTC). Generally, the detection markers of thisdisclosure can be used for cell identification, classification, andquantification.

The term “analysis marker” as used herein, refers to a biomarker that isused to describe a cell with respect to a cell biological or molecularbiological property of interest. For example, without limitation,analysis markers can describe certain aspects of a cellular genome(e.g., gene mutations (e.g., oncogene mutations), gene amplifications),transcriptome (gene expression profiles), proteome (protein expressionprofiles, post-translational protein modifications, intracellularprotein localization), secretome, metabolome (metabolic activity,including energy metabolism), lipidome (lipid profiles, lipid rafts) andthe like.

The terms “marker” and “biomarker” are used interchangeably throughoutthe disclosure. Such biomarkers include, but are not limited to,biological molecules comprising nucleotides, nucleic acids, nucleosides,amino acids, sugars, fatty acids, steroids, metabolites, peptides,polypeptides, proteins, carbohydrates, lipids, hormones, antibodies,regions of interest that serve as surrogates for biologicalmacromolecules and combinations thereof (e.g., glycoproteins,ribonucleoproteins, lipoproteins). The term also encompasses portions orfragments of a biological molecule, for example, peptide fragment of aprotein or polypeptide. In some embodiments, biomarkers (e.g., RCCanalysis markers) are disease marker (e.g., oncogenic mutations). Insome embodiments, biomarkers (e.g., RCC analysis markers) are used todistinguish and identify subpopulations of cells.

A person skilled in the art will appreciate that a number of methods canbe used to determine the presence or absence of a biomarker, includingmicroscopy based approaches, such as fluorescence microscopy orfluorescence scanning microscopy (see, e.g., Marrinucci D. et al., 2012,Phys. Biol. 9 016003; Nieva J. et al., 2012, Phys. Biol. 9 016004).Other approaches include mass spectrometry, gene expression analysis(e.g., gene-chips, Southern Blots, PCR, FISH) and antibody-basedapproaches, including immunofluorescence, immunohistochemistry,immunoassays (e.g., Western blots, enzyme-linked immunosorbent assay(ELISA), immunoprecipitation, radioimmunoassay, dot blotting, and FACS).In some embodiments, the methods of this disclosure are performed in anautomated or robotic fashion. In some embodiments, the signals frommultiple samples are detected simultaneously.

A person of skill in the art will further appreciate that the presenceor absence of biomarkers in a cell can be detected using any class ofmarker-specific binding reagents known in the art, including, e.g.,antibodies, aptamers, fusion proteins, such as fusion proteins includingprotein receptor or protein ligand components (e.g. CD 146, vWF, CD 31,CD 34, CD 105, or CD 45-binding receptors or ligands),biomarker-specific peptides, small molecule binders or nucleic acids(e.g., antisense oligonucleotides, hybridization probes).

In some embodiments, the presence or absence of vWF, CD 146, CD31, CD34, CD 105, CD 45 or a cytokeratin (e.g., cytokeratin 1, 4, 5, 6, 7, 8,10, 13, 18 or 19), or a combination thereof, is determined by anantibody. In some embodiments, the presence or absence of vWF and one ormore cytokeratins (e.g., cytokeratin 1, 4, 5, 6, 7, 8, 10, 13, 18 or 19)is determined by antibodies. In some embodiments, the presence orabsence of vWF, one or more cytokeratin (e.g., cytokeratin 1, 4, 5, 6,7, 8, 10, 13, 18 or 19) or CD 45 is determined by antibodies.

The antibodies of this disclosure bind specifically to a biomarker. Insome embodiments, the antibodies bind specifically to a single biomarker(e.g., cytokeratin 1). In other embodiments, the antibodies arepan-specific. Pan-specific antibodies of this disclosure can bindspecifically to one or more members of a biomarker family (e.g., one ormore members of the cytokeratin family, including cytokeratins 1, 4, 5,6, 7, 8, 10, 13, 18 and 19). The antibody can be any immunoglobulin orderivative thereof, whether natural or wholly or partially syntheticallyproduced. All antibody derivatives which maintain specific bindingability can also be used. The antibody has a binding domain that ishomologous or largely homologous to an immunoglobulin binding domain andcan be derived from natural sources, or partly or wholly syntheticallyproduced. The antibody can be a monoclonal or polyclonal antibody. Insome embodiments, the antibody is a single-chain antibody. In someembodiments, the antibody includes a single-chain antibody fragment. Insome embodiments, the antibody can be an antibody fragment including,but not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fdfragments. Due to their smaller size antibody fragments can offeradvantages over intact antibodies in certain applications. Alternativelyor additionally, the antibody can comprise multiple chains which arelinked together, for example, by disulfide linkages, and any functionalfragments obtained from such molecules, wherein such fragments retainspecific-binding properties of the parent antibody molecule. Those ofordinary skill in the art will appreciate that the antibody can beprovided in any of a variety of forms including, for example, humanized,partially humanized, chimeric, chimeric humanized, etc. The antibody canbe prepared using any suitable methods known in the art. For example,the antibody can be enzymatically or chemically produced byfragmentation of an intact antibody or it can be recombinantly producedfrom a gene encoding the partial antibody sequence.

A wide variety of detectable labels can be used for the direct orindirect detection of biomarkers. Suitable detectable labels include,but are not limited to, fluorescent dyes (e.g., fluorescein, fluoresceinisothiocyanate (FITC), Oregon Green™, rhodamine, Texas Red,tetrarhodamine isothiocynate (TRITC), Cy3, Cy5, Alexa Fluor® 647, AlexaFluor® 555, Alexa Fluor® 488), fluorescent protein markers (e.g., greenfluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g.,luciferase, horseradish peroxidase, alkaline phosphatase, etc.),nanoparticles, biotin, digoxigenin, metals, and the like.

In some embodiments, the biomarkers are fluorescent markers. In someembodiments, the biomarkers are immunofluorescent markers. In someembodiments, the biomarkers are fluorescence in situ hybridization(FISH) markers.

In some embodiments, the immunofluorescent markers are immunofluorescentanalysis markers. In some embodiments, the immunofluorescent markers areimmunofluorescent detection markers. In some embodiments, theimmunofluorescent detection markers are immunofluorescent RCC detectionmarkers. In some embodiments, the immunofluorescent RCC detectionmarkers are immunofluorescent CTC detection markers. In someembodiments, the immunofluorescent RCC detection markers areimmunofluorescent CEC detection markers.

In some embodiments, the immunofluorescent CTC detection markers includea cytokeratin (CK). Cytokeratins include, e.g., cytokeratin 1, 4, 5, 6,7, 8, 10, 13, 18 or 19. In some embodiments, the immunofluorescent CTCdetection marker is a plurality of cytokeratins, including two or moreof cytokeratins 1, 4, 5, 6, 7, 8, 10, 13, 18 or 19.

In some embodiments, the immunofluorescent CEC detection markers includeVon Willebrand factor (vWF), cluster of differentiation (CD) 31, CD 34,CD 105, CD 145 or CD 146.

In some cells the sample cell markers are immunofluorescent sample cellmarkers. In some embodiments, the immunofluorescent sample cell markersare specific for white blood cells (WBCs). In certain embodiments theimmunofluorescent sample cell markers comprise CD 45. In someembodiments, the methods include determining presence or absence or oneor more immunofluorescent sample cell detection markers in the nucleatedcells.

In some embodiments, the distinct immunofluorescent staining ofnucleated cells of a sample includes the presence or absence ofimmunofluorescent detection markers, such as immunofluorescent RCCdetection markers.

In some embodiments, the distinct immunofluorescent staining of CTCsincludes the presence of an immunofluorescent CTC detection marker, theabsence of an immunofluorescent CEC detection marker, and the absence ofan immunofluorescent sample cell detection marker. In some embodiments,the distinct immunofluorescent staining of CTCs includes positivestaining for CK, negative staining for vWF and negative staining forCD45 (CK⁺/vWF⁻/CD45⁻).

In some embodiments, the distinct immunofluorescent staining of CECsincludes the presence of an immunofluorescent CEC detection marker, theabsence of an immunofluorescent CTC detection marker and the absence ofan immunofluorescent sample cell detection marker. In some embodiments,the distinct immunofluorescent staining of CECs includes positivestaining for vWF, negative staining for CK and negative staining forCD45 (vWF⁺/CK⁻/CD45⁻).

In some embodiments, the distinct immunofluorescent staining of CTCmimics includes the presence of an immunofluorescent CTC detectionmarker, the presence of an immunofluorescent CEC detection marker, andthe absence of an immunofluorescent sample cell detection marker. Insome embodiments, the distinct immunofluorescent staining of CTC mimicsincludes positive staining for CK, positive staining for vWF andnegative staining for CD45 (CK⁺/vWF⁺/CD45⁻).

In some embodiments, the distinct immunofluorescent staining of CTCcandidates includes the presence of an immunofluorescent CTC detectionmarker, the absence of an immunofluorescent CEC detection marker, andthe absence of an immunofluorescent sample cell detection marker. Inother embodiments, the distinct staining of CTC candidates includes thepresence of an immunofluorescent CTC detection marker, the presence ofan immunofluorescent CEC detection marker, and the absence of animmunofluorescent sample cell detection marker. In some embodiments, thedistinct immunofluorescent staining of CTC candidates includes positivestaining for CK and negative staining for CD45 (CK⁺/CD45).

In some embodiments, the distinct immunofluorescent staining of a samplecell includes the presence of an immunofluorescent sample cell detectionmarker, the absence of an immunofluorescent CEC detection marker and theabsence of an immunofluorescent CTC detection marker.

In some embodiments, the distinct immunofluorescent staining of a CEC,CTC, CTC mimic, CTC candidate or sample cell includes distinctintracellular staining patterns for an immunofluorescent CEC detectionmarker, an immunofluorescent CTC detection marker, or animmunofluorescent sample cell detection marker. For example, theintracellular staining for an immunofluorescent marker of thisdisclosure can be distinctly diffuse, punctuate, cytoplasmic, nuclear ormembrane bound.

In some embodiments, determining presence or absence of animmunofluorescent RCC detection marker comprises comparing the distinctimmunofluorescent staining of RCCs with the distinct immunofluorescentstaining of WBCs.

In some embodiments, determining presence or absence of animmunofluorescent CTC detection marker includes comparing the distinctimmunofluorescent staining of CTC candidates with the distinctimmunofluorescent staining of a sample cell. In some embodiments,determining presence or absence of an immunofluorescent CTC detectionmarker includes comparing the distinct immunofluorescent staining of CTCcandidates with the distinct immunofluorescent staining of WBCs.

In some embodiments, determining presence or absence of animmunofluorescent CEC detection marker includes comparing the distinctimmunofluorescent staining of CEC candidates with the distinctimmunofluorescent staining of a sample cell. In some embodiments,determining the presence or absence of an immunofluorescent CECdetection marker includes comparing the distinct immunofluorescentstaining of CTC candidates with the distinct immunofluorescent stainingof WBCs.

In some embodiments, the morphological characteristics include nucleussize, nucleus shape, cell size, cell shape, and nuclear-to-cytoplasmicratio. In some embodiments, assessing the morphology of RCCs includesassessing the RCCs by nuclear detail, nuclear contour, presence orabsence of nucleoli, quality of cytoplasm, quantity of cytoplasm, orimmunofluorescent staining patterns. In some embodiments, the methodfurther comprises assessing the aggregation characteristics of RCCs.

A person of ordinary skill in the art understands that the morphologicalcharacteristics of this disclosure can include any feature, property,characteristic or aspect of a cell that can be determined and correlatedwith the detection of RCCs.

The methods of this disclosure can be performed with any microscopicmethod known in the art. In some embodiments, the method is performed byfluorescent scanning microscopy. In some embodiments the microscopicmethod provides high-resolution images of RCCs and their surroundingWBCs (see, e.g., Marrinucci D. et al., 2012, Phys. Biol. 9 016003; NievaJ. et al., 2012, Phys. Biol. 9 016004). In some embodiments, a slidecoated with a monolayer of nucleated cells from a sample, such as anon-enriched blood sample, is scanned by a fluorescent scanningmicroscope and the fluorescence intensities from immunofluorescentdetection markers and nuclear stains are recorded. The scanned imagesare analyzed to determine the presence or absence of immunofluorescentdetection markers and to assess the morphology of the nucleated cells,including RCCs. In some embodiments, microscopic data collection andanalysis is conducted in an automated manner.

In some embodiments, the microscopic field contains RCCs and WBCs. Insome embodiments, the microscopic field shows at least 1, 5, 10, 20, 50,or 100 RCCs. In some embodiments, the microscopic field shows at least10, 25, 50, 100, 250, 500, or 1,000 fold more WBCs than RCCs. In certainembodiments, the microscopic field shows RCCs, wherein each RCC issurrounded by at least 10, 50, 100, 150, 200, 250, 500, 1,000 or moreWBCs.

In certain embodiments, the microscopy provides a field of viewcomprising more than 2, 5, 10, 20, 30, 40 or 50 RCCs, wherein each RCCis surrounded by more than 10, 50, 100, 150 or 200 WBCs. In someembodiments, the microscopy provides a field of view comprising morethan 10 RCCs, wherein each RCC is surrounded by more than 200 WBCs.

In some embodiments, a biomarker is considered “present” in a cell if itis detectable above the background signal and noise of the respectivedetection method used (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higherthan the background; 2σ or 3σ over background). In some embodiments, abiomarker is considered “absent” if it is not detectable above thebackground noise of the detection method used (e.g., <1.5-fold or<2.0-fold higher than the background signal; <1.5σ or <2.0σ overbackground).

In some embodiments, the presence or absence of immunofluorescentmarkers in nucleated cells is determined by selecting the exposure timesduring the fluorescence scanning process such that all immunofluorescentmarkers achieve a pre-set level of fluorescence on the WBCs in the fieldof view. Under these conditions, immunofluorescent RCC detectionmarkers, for example, are visible on the WBCs as background signals withfixed heights, even though the respective immunofluorescent RCCdetection markers are not present in WBCs. Moreover, WBC-specificimmunofluorescent sample cell detection markers are visible on RCCs asbackground signals with fixed heights, even though the markers are notpresent in RCCs.

A cell is considered positive for an immunofluorescent marker (i.e., themarker is considered present) if its fluorescent signal for therespective marker is significantly higher than the fixed backgroundsignal (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higher than thebackground; 2σ or 3σ over background). For example, a nucleated cell isconsidered CD 45-positive (CD 45⁺) if its fluorescent signal for CD 45is significantly higher than the background signal. A cell is considerednegative for an immunofluorescent marker (i.e., the marker is consideredabsent) if the cell's fluorescence signal for the respective marker isnot significantly higher than the background signal or noise (e.g.,<1.5-fold or <2.0-fold higher than the background signal; e.g., <1.5σ or<2.0σ over background).

The relative expression levels of an immunofluorescent RCC detectionmarker can be expressed by comparing the fluorescence signal of a cellthat is positive for the respective marker (i.e., a CTC, CTC candidate,CTC mimic or CEC) with the corresponding fluorescence signal ofsurrounding cells that are negative for the immunofluorescent RCCdetection marker (e.g., a WBC). For example, the relative expression ofthe CTC marker cytokeratin on a given CTC candidate is >5 if thefluorescence signal for cytokeratin on the cell is >5-fold higher than,e.g., the average or median fluorescence signal of surrounding WBCs.

A cell is considered a nucleated cell if it shows a fluorescence signalfor a nuclear stain (e.g., DAPI) that is significantly higher than thebackground signal or noise, e.g., as detected for a non-nucleatedplatelet cell or for representative cell-free areas on a microscopeslide.

In some embodiments, determining the presence of an immunofluorescentCTC detection marker in nucleated cells includes identifying nucleatedcells having a relative expression of the CTC detection markerof >2, >3, >4, >5, >6, >7, >8, >9 or >10. In some embodiments,determining the presence of CK in nucleated cells includes identifyingnucleated cells having a relative CK expression of >3.

In some embodiments, determining the presence of an immunofluorescentCEC detection marker in CTC candidates includes identifying CTCcandidates having a relative expression of the CEC detection markerof >2, >3, >4, >5, >6, >7, >8, >9 or >10. In some embodiments,determining the presence of vWF in CTC candidate includes identifyingCTC candidate cells having a relative vWF expression of >6.

In some embodiments, the morphological assessment of a nucleated cell,such as the determination of its size or shape, is based on thefluorescence signals of an immunofluorescent detection marker (see,e.g., Marrinucci D. et al., 2012, Phys. Biol. 9 016003; Nieva J. et al.,2012, Phys. Biol. 9 016004).

In some embodiments, the RCCs are morphologically distinct from thesurrounding nucleated cells, such as WBCs. In some embodiments,assessing the morphology of the nucleated cells comprises comparing themorphological characteristics of the RCC with the morphologicalcharacteristics of surrounding WBCs.

In some embodiments, the CTCs, CTC mimics, CTC candidates and CECs aremorphologically distinct from the surrounding WBCs. In some embodiments,assessing the morphology of the CTC candidate comprises comparing themorphological characteristics of the CTC candidate with themorphological characteristics of surrounding WBCs.

In some embodiments, the CTCs, CTC mimics, CTC candidates and CECs aremorphologically distinct from each other. In some embodiments, assessingthe morphology of the CTC candidate comprises comparing themorphological characteristics of the CTC candidate with themorphological characteristics of a CTC. In some embodiments, assessingthe morphology of the CTC candidate comprises comparing themorphological characteristics of the CTC candidate with themorphological characteristics of a CEC.

Morphological features shared between CTCs and CTC mimics include, forexample and without limitation, the presence of distinct and intactnuclei, the presence of nuclei with irregular shapes, the presence ofcondensed chromatin, a nuclear area that is larger than the nuclear areaof WBCs, a cytoplasmic area that is larger than the cytoplasmic area ofWBCs, a higher cytoplasmic-to-nuclear ratio relative to WBCs, thepresence of aggregates of two or more cytokeratin positive (CK⁺) cells,or combinations thereof.

Morphological features shared between CECs and CTC mimics include, forexample and without limitation, the presence of nuclei with irregularshapes, the presence of elongated nuclei, the presence of an elongatedcytoplasm, a nuclear area that is larger than the nuclear area of WBCs,a cytoplasmic area that is larger than the cytoplasmic area of WBCs, ahigher cytoplasmic-to-nuclear ratio relative to WBCs, the presence ofaggregates of two or more cytokeratin positive (vWF⁺) cells, orcombination thereof.

In some embodiments, the (average or mean) nuclear area of RCCs in amicroscopic field of view is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% or 50% greater than the nuclear area of WBCs.

In some embodiments, the (average or mean) cytoplasmic area of RCCs in amicroscopic field of view is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% or 50% greater than the cytoplasmic area of WBCs.

In some embodiments, the (average or mean) cytoplasmic-to-nuclear ratioof RCCs in a microscopic field of view is at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50% greater than the cytoplasmic-to-nuclearratio of WBCs.

In some embodiments (also referred to as “high-definition (HD)”-RCCAssay), the comparison of an RCC (i.e., a target cells of interest) withsurrounding WBCs (i.e., negative control cells) improves the performanceof the method, e.g., by increasing the accuracy, specificity, orsensitivity of the method, relative to a method wherein no suchcomparison is performed. In some embodiments, RCCs are compared withsurrounding WBCs when determining the presence or absence of animmunofluorescent RCC detection marker. In some embodiments, RCCs arecompared with surrounding WBCs when assessing the morphology ofnucleated cells. In some embodiments, RCCs are compared with surroundingWBCs when determining the presence or absence of an immunofluorescentRCC detection marker and when assessing the morphology of nucleatedcells.

In some embodiments, assessing the morphology of the nucleated cellsincludes assessing the morphology of RCC aggregates. In someembodiments, assessing the morphology of RCC aggregates includesquantifying the RCC aggregates in the blood sample. In some embodiments,assessing the morphology of RCC aggregates includes assessing thepercent of detected RCCs that are in an aggregated from. In someembodiments, assessing the morphology of RCC aggregates includesquantifying the average or mean number of cells per aggregate in asample.

In some embodiments, the methods are used to calculate the concentrationof RCCs in a sample (e.g., in [RCC/ml]). For example, CTCs are detectedin a human blood sample according to the methods of this disclosure.Next, the ratio of CTCs to total nucleated cells (i.e., CTCs, CTCcandidates, CTC mimics, CECs plus sample cells, such as WBCs) isdetermined for a field of vision. Then, the CTC mimic/total nuclear cellratio is multiplied by the concentration of total nucleated cells in ablood sample (e.g., as determined using a standard automated cellcounter) to calculate the concentration of CTC mimics in the bloodsample.

According to the methods of this disclosure, the RCCs can be furtheranalyzed after they were detected, and optionally quantified, by amethod of this disclosure.

In some embodiments, the methods of this disclosure include quenchingthe staining of the one or more RCC detection markers comprisingcontacting the RCCs with a quenching buffer, wherein the staining isquenched by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 99%.

In some embodiments, the methods of this disclosure include quenchingthe immunofluorescence of the one or more immunofluorescent RCCdetection markers comprising contacting the RCCs with a quenchingbuffer, wherein the immunofluorescence is quenched by more than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

In some embodiments, the quenching buffer contains a chaotropic agent.The term “chaotropic agent”, as used herein, refers to a substance thatcan disrupt the secondary or tertiary structure of biologicalmacromolecules, such as proteins and nucleic acids (e.g., DNA, RNA) ordissolve lipid bilayers, such as plasma membranes. Chaotropic agentsinclude, without limitation, chaotropic salts (e.g., guanidiniumchloride (guanidine chloride), lithium perchlorate, lithium acetate,magnesium chloride, sodium dodecyl sulfate), chaotropic solvents (e.g.,butanol, ethanol), or uncharged, solid chaotropes (e.g., urea,thiourea).

In some embodiments the concentration of the chaotropic agent is lessthan 10 M, less than 8 M, less than 6 M, less than 4 M, less than 2 M,less than 1 M, or less than 0.5 M. In some embodiments, theconcentration of the chaotropic agent is at least 0.5M, at least 1 M, atleast 2M, at least 4M, at least 6M or at least 8M.

In some embodiments, the quenching buffer contains a chaotrophic salt.In some embodiments, the quenching buffer contains guanidine or aguanidine salt. In some embodiments, the quenching buffer containsguanidinium thiocyanate (guanidine thiocyanate) or guanidinium chloride(guanidine hydrochloride).

In some embodiments, the RCCs are contacted with the quenching bufferfor a period of time of more than 1 minute, more than 2 minutes, morethan 3 minutes, more than 4 minutes, more than 5 minutes, more than 6minutes, more than 7 minutes, more than 8 minutes, more than 9 minutes,more than 10 minutes, more than 15 minutes or more than 20 minutes. Insome embodiments, the RCCs are contacted with the quenching buffer for aperiod of time of less than 1 minute, less than 45 seconds, less than 30seconds, less than 15 seconds, less than 10 second or less than 5seconds.

In some embodiments, the RCCs are contacted with the quenching buffer ata temperature of less than 37° C., less than 34° C., less than 30° C.,less than 25° C., less than 20° C., less than 15° C., less than 10° C.,less than 5° C., or less than 1° C. In some embodiments, RCCs arecontacted with the quenching buffer at a temperature of about 4° C.

In some embodiments, the RCCs are detected in a non-enriched bloodsample placed on a solid support (e.g., on a glass slide). In someembodiments, a certain fraction of RCCs detected in the HD-RCC assay iswashed off the solid support during incubation with the quenchingbuffer. These cells are physically unavailable for further analysis.

In some embodiments, more than 50%, more than 55%, more than 60%, morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 95% or more than 99% of detected RCCs that werepresent on the solid support prior to incubation with quenching bufferare retained on the solid support after incubation with quenchingbuffer.

In some embodiments, more than 50%, more than 55%, more than 60%, morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 95% or more than 99% of RCCs that were detectedin the blood sample by a method of this disclosure are physicallypresent for further analysis after incubation with quenching buffer.

In some embodiments, more than 25%, more than 30%, more than 35%, morethan 40%, more than 45%, more than 50%, more than 55%, more than 60%,more than 65%, more than 70%, more than 75%, more than 80%, more than85%, more than 90%, more than 95%, or more than 99% of RCCs detected in(a) in a method of this disclosure are retained in (c) in a method ofthis disclosure.

In some embodiments, the methods include analyzing the detected RCCs bydetermining presence or absence of one or more fluorescent RCC analysismarkers. In some embodiments, the RCC analysis markers are present inmore than 25%, more than 30%, more than 35%, more than 40%, more than45%, more than 50%, more than 55%, more than 60%, more than 65%, morethan 70%, more than 75%, more than 80%, more than 85%, more than 90%,more than 95% or more than 99% of RCCs analyzed in (c) in a method ofthis disclosure.

In some embodiments, determining presence or absence of the fluorescentRCC analysis markers includes comparing the distinct fluorescentstaining of RCCs with the distinct fluorescent staining of WBCs.

The RCC analysis marker can include any molecular probe that indicatesthe cell biological or molecular biological status of an RCC. The cellbiological or molecular biological status of an RCC can include, withoutlimitation, cell morphological characteristics, cellular dynamics (e.g.,cell motility, adhesion to extracellular matrix substrates),intracellular localization or structural characteristics (e.g.,intracellular localization of organelles, biomolecules; formation andlocalization of biomolecular assemblies, such as lipid rafts), metaboliccharacteristics (e.g., energy metabolism, intracellular signaling),genomic characteristics (e.g., gene expression, gene mutations, mRNAsplicing) or proteomic characteristics (protein expression,localization, post-translational modification, secretome profile).

In some embodiments, the RCC analysis marker includes a genetic mutation(e.g., a gene deletion, duplication, amplification, translocation,point-mutation). In some embodiments, the RCC analysis marker includesthe elevated expression of a gene of interest (e.g., an oncogene). Insome embodiments, the RCC analysis marker includes the reducedexpression of a gene of interest (e.g., a tumor suppressor gene). Insome embodiments, the RCC analysis marker includes the elevatedexpression of an mRNA of interest. In some embodiments, the RCC analysismarker includes the elevated expression of a protein of interest (e.g.,HER2, Bcl-2). In some embodiments, the RCC analysis marker includes aspecific intracellular localization of a protein of interest (e.g.,nuclear localization, cytoplasmic localization). In some embodiments,the RCC analysis marker includes a post-translational proteinmodification (e.g., phosphorylation, methylation).

In some embodiments, the RCC analysis marker is a genetic mutation,including a gene translocation, a gene inversion, a gene amplification,a gene deletion, gene aneuploidy or chromosomal aneuploidy.

In some embodiments, the RCC analysis marker is an oncogene. Oncogenesinclude, without limitation, PTEN, ALK, PIK3CA, MET, ROS, RET, HER2,ERG, AURKA, BRCA 1, BRCA 2, P53, RAS, RAF, EGFR, HER2, WNT, MYC, FAS,TRK, CDK, SRC, SYK, BTK and ABL.

In some embodiments, the RCC analysis marker is a positive controlmarker. A positive control marker can be any molecular or cellularmarker expected to be present in essentially every cell in a RCCpopulation of interest, e.g., in every RCC in a microscopic field ofview. In some embodiments, the positive control marker is a chromosomalmarker (e.g., a marker for human chromosomes 10, 15, 5, 3 and the like).In some embodiments, the positive control marker is a telomere marker.

In some embodiments, the determination of the presence or absence of apositive control marker is a measure for whether a cell of interest,e.g., an RCC in a microscopic field of view, is amenable to furtheranalysis following detection of the RCC in an HD-RCC assay of thisdisclosure. Without wishing to be bound by theory, it is believed thatRCCs that were successfully detected in (a) in a method of thisdisclosure, but in which a positive control maker cannot be detected in(c) in a method of this disclosure were damaged during the quenchingstep (b) such that these RCC are not amenable to further molecular orcellular analysis. By contrast, RCCs that were successfully identifiedand in which a positive control marker can be detected are amenable tofurther analysis.

In some embodiments, the positive control marker is determined to bepresent in more than 25%, more than 30%, more than 35%, more than 40%,more than 45%, more than 50%, more than 55%, more than 60%, more than65%, more than 70%, more than 75%, more than 80%, more than 85%, morethan 90%, more than 95%, or more than 99% of the detected RCCs analyzedin (c).

The presence or absence of an RCC analysis marker in a cell can bedetected using any class of marker-specific binding reagents (RCCanalysis probes) known in the art, including, e.g., antibodies,aptamers, fusion proteins, such as fusion proteins including proteinreceptor or protein ligand components, marker-specific peptides, smallmolecule binders or nucleic acids (e.g., antisense oligonucleotides,hybridization probes).

The RCC analysis markers can be detected by methods such as fluorescencescanning microscopy, mass spectrometry, gene-chips, protein-chips,immunocytochemistry, whole genome sequencing and the like. In someembodiments, presence or absence of an RCC marker is detected bydetecting gene copy number variants, by exome sequencing, by themutational analysis of biomarker genes, or by polymerase chain reaction(PCR).

In some embodiments, the RCC analysis marker is a plurality of RCCanalysis markers.

In some embodiments, the RCC analysis marker is a fluorescent analysismarker.

In some embodiments, the RCC analysis marker is a fluorescence in situhybridization (FISH) marker. In some embodiments, the FISH marker is achromosomal marker for genetic abnormalities, including, withoutlimitation, gene fusions, aneuploidy and loss of chromosomal regions. Insome embodiments, the FISH marker is a chromosomal marker for geneticmutations, including gene translocation, gene amplification and genedeletions. In some embodiments, the FISH marker is a positive controlmarker.

In some embodiments, the presence or absence of a FISH marker isdetected using a FISH probe. In some embodiments, FISH probes comprisesynthetic DNA oligonucleotides linked to a fluorescent dye. A skilledartisan will recognize that many fluorescent dyes for FISH probes arewell known in the art, including, without limitation, SpectrumOrange™,SpectrumGreen™ and SpectrumAqua™. In some embodiments, the FISH-probebinds to a DNA molecule (DNA-FISH). In some embodiments, the FISH-probebinds to an RNA molecule (RNA-FISH).

FISH probes include, without limitation, locus specific probes, each ofwhich binds to a particular region of a chromosome, alphoid orcentromeric repeat probes, which are generated from repetitive sequencesfound on the middle of each chromosome, and whole chromosome probes,which include collections of smaller probes, each of which binds to adifferent sequence along the length of a given chromosome. Other FISHprobes include, without limitation, whole chromosome painting probes(WPP), chromosome arm painting probes (APP), chromosome terminal bandpainting probes (TPP), chromosome enumeration probes (CEP), chromosomesubtelomere probes (CSP) and chromosome loci specific probes (CLP), alsocommonly called LSI (Locus specific identifier) probes.

FISH probes can be used alone or in combination with other FISH probesor with other RCC analysis probes. Combinations of FISH probes caninclude more than 2 probes, more than 3 probes, more than 4 probes, morethan 5 probes, more than 6 probes, more than 7 probes, more than 8probes, more than 9 probes, more than 10 probes, more than 15 probes,more than 20 probes, more than 25 probes, more than 50 probes, more than75 probes or more than 100 probes. In some embodiments, combinations ofFISH probes include 1-color probes, 2-color probes, 3-color probes or4-color probes.

In some embodiments, the methods further comprise contacting the RCCswith a FISH probe. In some embodiments, the RCCs are contacted with theFISH probe for more than 1 minute, more than 2 minutes, more than 3minutes, more than 4 minutes, more than 5 minutes, more than 6 minutes,more than 7 minutes, more than 8 minutes, more than 9 minutes, more than10 minutes, more than 15 minutes or more than 20 minutes.

In some embodiments, the RCCs are contacted with the FISH probe at atemperature of more than about 35° C., more than about 37° C., more thanabout 40° C., more than about 42° C., more than about 44° C. or morethan about 46° C.

In some embodiments, analyzing the detected RCCs further comprisesassessing the morphology of the detected RCCs. In some embodiments, thetreatment of RCCs with quenching buffer does not significantly alter themorphological characteristics of the RCCs.

In some aspects, the methods of this disclosure are used to detect,quantify and characterize RCCs in non-enriched blood samples from humanpatients.

In some embodiments, the non-enriched blood sample is a plurality ofnon-enriched blood samples. In some embodiments, the RCC analysismarkers are present in more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 85%, 90%, 95% or 99% of analyzed RCCs in more than 80%,85%, 90%, 95% or 99% of blood samples in the plurality of blood samples.In some embodiments, the plurality of non-enriched blood samples wasobtained from a plurality of patients.

From the foregoing description, it will be apparent that variations andmodifications can be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

The following examples are provided by way of illustration, notlimitation.

EXAMPLES Example 1: Identification, Quantification and Characterizationof CTCs in Non-Enriched Blood Samples from Human Cancer Patients

First, blood samples were obtained from three confirmed non-small celllung cancer (NSCLC) patients. CTC candidates were identified in eachsample as described, e.g., by Marrinucci et al. (2012) Phys Biol 9(1)016003 or Nieva et al. (2012) Phys Biol 9(1) 016004.

Briefly, blood samples underwent red blood cell lysis followed bymonolayer preparation of all nucleated cells on custom glass substrates.After paraformaldehyde (PFA) fixation and methanol permeabilization,cells were incubated with pan anti-cytokeratin antibodies recognizingcytokeratins 1, 4, 5, 6, 7, 8, 10, 13, 18 and 19 and a preconjugatedanti-CD45 antibody followed by incubation with an Alexa™ 555-conjugatedsecondary antibody and DAPI as a nuclear stain. All nucleated cells inthe specimen were imaged in multiple fluorescent channels to producehigh-quality and high-resolution digital images that retain finecytologic detail of nuclear contour and cytoplasmic distribution. Cellsthat were both cytokeratin positive (CK⁺) and CD45 negative (CD45⁻) wereidentified using custom computer algorithms and then subjected tomorphological analysis (e.g., analysis of their nuclear-to-cytoplasmicratio). Cells were evaluated by direct review of captured microscopicimages and classified as a CTC candidate based on cell morphology (e.g.,their low nuclear-to-cytoplasmic ratios) and immunophenotype (e.g.,CK⁺/CD45⁻).

Example 2: FISH-Analysis of CTCs in Non-Enriched Blood Samples fromHuman Cancer Patients

This example demonstrates that CTCs can be further analyzed with respectto their molecular or cell biology after they were identified in anHD-CTC assay. This example further demonstrates that an effectivequenching buffer was identified that effectively quenched theimmunofluorescent staining (e.g., the staining of immunofluorescent CTCdetection markers) while maintaining the identified CTCs in a conditionallowing their subsequent analysis in an fluorescent in situhybridization (FISH) assay.

FISH Protocol Following CTC Identification by HD-CTC Assay

Following the HD-CTC assay protocol described above, the non-enrichedblood samples were further processed as follows.

Cover slips were removed from the microscope slides and the slides wereplaced into a quenching buffer. Quenching buffers, incubation times andincubation temperatures were as further described below. The slides werethen incubated for another 5 min in 5% formaldehyde in PBS. Next, theslides were transferred to 70% ethanol in water, incubated for 2 min,transferred into 85% ethanol in water, incubated for another 2 min,transferred into 100% ethanol and incubated for another 2 min. Thebackside and sides of the slides were wiped with a delicate task wipeand the slides were allowed to air dry. Then, 20 μl of FISH probesolution (containing, e.g., a target-gene probe, including PTEN or ERGprobes, or a control probe, including centromere probes) inhybridization buffer was applied in an even line across a coverslip, thecoverslip was placed probe side down onto the slide. The probe solutionwas allowed to spread to the borders of the cover slip and thecoverslips were gently pushed to eliminate air bubbles. The borders werethen sealed with rubber cement. A 10 min incubation at 83° C. (allowingfor DNA denaturation) was followed by a 1-24 hour incubation at 37° C.(allowing for DNA-FISH probe hybridization). The coverslips were thenremoved and the slides were first placed in 0.4×SSC/0.3% Igepalsolution, pH 7 for 2 minutes and afterwards transferred to 2×SSC/0.1%Igepal solution, pH 7 for 1-10 minutes. The slides were removed, theDAPI counterstain was applied and the edges of the coverslip were sealedwith nail polish. Finally, the slides were analyzed by fluorescencescanning microscopy.

Comparison of Quenching Buffers

In exemplary experiments, the identification of CTCs in non-enrichedblood samples involved the immunofluorescent detection of cytokeratin(CK). Surprisingly, incubation of CTC samples with a 4 M guanidiniumthiocyanate (GdnSCN) quenching buffer resulted in an effective reductionof CK immunofluorescence while largely maintaining the CTC cellmorphology (e.g., cell shapes and sizes, nuclei shapes and sizes). See,e.g., FIG. 1.

In typical experiments, quenching buffer incubation times around 5-10minutes were found to be sufficient to achieve the reduction of CKimmunofluorescence. Moreover, quenching buffer incubation temperaturesbetween 4° C. and room temperature resulted in an efficient reduction ofCK immunofluorescence. GdnSCN concentrations as low as 2 M were found tobe sufficient to effectively reduce CK immunofluorescence. See, e.g.,FIG. 4.

Quenching of CK immunofluorescence was found to be more efficient onCTCs having lower relative CK expression than in cells having higherrelative CK expression. Generally, GdnSCN buffers were found to quenchCK immunofluorescence by at least about 75%. In some experiments, theimmunofluorescence was quenched by up to 90%, up to 99% or more.

Surprisingly, the incubation with GdnSCN buffers resulted in theretention of the majority of previously identified CTCs on themicroscope slides. Generally, more than 60% of previously identifiedCTCs were retained. In some experiments, more than 70%, 80% and even upto 95% of previously identified CTCs were retained.

Typically, more than 85% of the retained CTCs showed signals forpositive control FISH markers, such as the chromosome 10 centromeremarker. In some experiments, more than 70%, 80% and even more than 95%of previously identified CTCs showed positive signals for positivecontrol FISH markers.

The surprising activity of the GdnSCN buffer was found to depend on thepresence of the chaotripic agent guanidinium. For example, sodiumthiocyanate (NaSCN) buffers were found not to quench CKimmunofluorescence. See, e.g., FIG. 3.

Additionally, many other buffers that are commonly used in molecular orcell biology protocols were either found to be ineffective quenchers ofCK immunofluorescence (see, e.g., FIGS. 1, 2, 5, 6 and 7) or were foundto destroy CTC cell morphology (see, e.g., FIG. 5, center column).

Ineffective buffers included, Sodium Thiocyanate (NaSCN), ThermoScientific Restore™ Western Blot Stripping Buffer, Glycine/SDS at pH 2,Glycine/SDS at pH 4, Glycine/SDS at pH 7, Glycine/SDS at pH 10, andother Tris-buffers containing combinations of SDS, Tris,Betamercaptoethanol or dithiotreitol (DTT).

Results of an exemplary FISH experiment conducted on the cells of anon-enriched blood sample following the identification of CTCs in anHD-CTC assay and the quenching of CK-immunofluorescence with 4M GdnSCNbuffers are shown in FIG. 8.

In summary, guanidinium-containing buffers were found to effectivelyquench CTCs' immunofluorescence following their detection in HD-CTCassays while retaining the majority of identified CTCs available forfurther analysis and while maintaining CTC cell morphology.

Although the disclosure has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the disclosure. It should be understood that variousmodifications can be made without departing from the spirit of thedisclosure. Accordingly, the disclosure is limited only by the followingclaims.

What is claimed:
 1. A method for analyzing rare circulating cells (RCCs)in a non-enriched blood sample, comprising: (a) detecting RCCs in thenon-enriched blood sample, comprising i) determining presence or absenceof one or more immunofluorescent RCC detection markers in nucleatedcells in the non-enriched blood sample, and ii) assessing the morphologyof the nucleated cells, wherein RCCs are detected among the nucleatedcells based on a combination of distinct immunofluorescent staining andmorphological characteristics; (b) quenching the immunofluorescence ofthe one or more immunofluorescent RCC detection markers comprisingcontacting the RCCs with a quenching buffer, wherein the quenchingbuffer comprises a chaotropic salt and wherein the immunofluorescence isquenched by more than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or99.99%; and (c) analyzing the detected RCCs, comprising determiningpresence or absence of one or more fluorescent RCC analysis markers,wherein analyzing the detected RCCs comprises assessing the morphologyof the detected RCCs.
 2. The method of claim 1, wherein the fluorescentRCC analysis markers are fluorescence in situ hybridization (FISH)markers.
 3. The method of claim 1, wherein more than 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of RCCsdetected in (a) are retained in (c).
 4. The method of claim 1, whereinthe fluorescent RCC analysis markers are positive control markers. 5.The method of claim 4, wherein the positive control markers are presentin more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% of RCCs analyzed in (c).
 6. The method of claim 4,wherein the positive control markers are chromosomal markers.
 7. Themethod of claim 4, wherein the positive control markers are centromeremarkers or telomere markers.
 8. The method of claim 1, wherein the RCCanalysis markers are genetic mutations selected from the groupconsisting of gene translocation, gene amplification, gene deletion,gene aneuploidy and chromosomal aneuploidy.
 9. The method of claim 1,wherein the RCCs are circulating tumor cells (CTCs).
 10. The method ofclaim 1, wherein the RCCs are circulating epithelial cells (CECs). 11.The method of claim 1, wherein the RCCs are CTC mimics.
 12. The methodof claim 1, wherein the RCCs are CTC candidates.
 13. The method of claim1, wherein the quenching buffer comprises a chaotropic agent.
 14. Themethod of claim 13, wherein the concentration of the chaotropic agent isat least 2M, 3M or 4M.
 15. The method of claim 1, wherein the quenchingbuffer comprises guanidine or a guanidinium salt.
 16. The method ofclaim 1, wherein the quenching buffer comprises guanidinium thiocyanate(guanidine thiocyanate) or guanidinium chloride (guanidinehydrochloride).
 17. The method of claim 1, wherein the RCCs werecontacted with the quenching buffer for a period of time of more than 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes or more than 20minutes.
 18. The method of claim 1, wherein the analyzed RCCs compriseRCC aggregates.